The root system of potato (Solanum tuberosum L. cv. Favorita) plants was treated with different O₂ and CO₂ concentrations for 35 d in aeroponic culture. Under 21 or 5 % O₂ in the root zones, the thickness of leaves and palisade parenchyma significantly increased at 3 600 μmol(CO₂) mol^-1 in the root zone, compared with CO₂ concentration 380 μmol mol^-1 or low CO₂ concentration (100 μmol mol^-1). In addition, smaller cells of palisade tissue, more intercellular air spaces and partially two layers of palisade cells were observed in the leaves with root-zone CO₂ enrichment. Furthermore, there was a significant increase in the size of chloroplasts and starch grains, and the number of starch grains per chloroplast due to elevated CO₂ only under 21 % O₂. In addition, a significant decline in the thickness of grana and the number of lamellas, but no significant differences in the number of grana per chloroplast were observed under elevated CO₂ concentration. The accumulation of starch grains in the chloroplast under elevated CO₂ concentration could change the arrangement of grana thylakoids and consequently inhibited the absorption of sun radiation and photosynthesis of potato plants.

Leucoanthocyanidin reductase (LAR) converts leucoanthocyanidin to (+)-catechin, a precursor of proanthocyanidins abundant in Japanese persimmon (Diospyros kaki Thunb.) fruits. A putative LAR gene (DkLAR) was isolated by rapid amplification of cDNA ends from young fruits. The full-length cDNA of DkLAR gene was 1 356 bp long and encoded an open reading frame of 349 residues. The deduced DkLAR protein was closely related to the homolog in other plant species. The expression of the DkLAR gene in Chinese pollination-constant non-astringent (PCNA) genotype was coincident with the tannin cell development, but was not in Japanese PCNA and Chinese pollination-variant astringent (PCA) genotypes.

Exposure of pea plants to high irradiance (HI) for 60 min caused a reversible photoinhibition as shown by changes in variable to maximum fluorescence ratio (F_v/F_m). A significant decline in F_m was observed in leaves from both pea cultivars subjected to HI, the decrease being higher in JI281 than in JI399 plants. The values recovered during the post-stress period in both cultivars. In both cultivars, minimal fluorescence (F₀) increased under HI, but in cultivar JI399 F₀ recovered to initial value during the post-stress period. The expression of antioxidant enzyme genes was higher in JI399 than in JI281, both in control and stressed plants. In JI281, after 60 min of HI, an induction of the transcripts of CAT, chlMDHAR, cytAPX and cytCu,Zn-SOD was observed, whereas there was a slight increase in PHGPX, stAPX and chlCu,Zn-SOD mRNAs. After 24 h of the recovery period, the induction of some transcripts was not maintained (CAT, cytAPX and cytCu,Zn-SOD), whereas the induction of others was maintained (PHGPX and chlCu,ZnSOD) or even increased (cytGR, stAPX and chlMDHAR). In JI399, CAT and cytAPX were increased strongly after 60 min of HI, and slight increases were observed in cytGR, chlGR and chlMDHAR. In the post-stress period the expression of stAPX, cytGR and chlMDHAR was even slightly higher than after 60 min of HI, however, expression of CAT, cytAPX, cytCu,ZnSOD, chlCu,ZnSOD and chlGR decreased.

The effects of two-day dark treatment, applied to whole plants or to individual organs, on the photosynthetic apparatus in cotyledons and first rosette leaves of young Arabidopsis thaliana plants were studied. Darkness affected the individually darkened pair of cotyledons as well as the cotyledons of whole darkened plants (DP) in a similar manner as revealed by the significant decrease in the actual yield of photosystem 2 electron transport and the down-regulation of the psaB and rbcL transcript levels. However, cotyledons and rosette leaves responded differently to darkness with respect to the non-photochemical quenching (NPQ) and the non-regulated energy dissipation (Φ_NO), indicating different capacity for photoprotection depending on the type of the applied dark treatment. Besides, the expression of the genes for the two plastid proteases FtsH5 and Deg1 involved in D₁ protein degradation was inhibited in both leaf organs, suggesting that these proteases function mainly under irradiance. Upon re-irradiation, dark-treated cotyledons recovered from the applied stress and during further senescence the changes in the photosynthetic parameters and the mRNA levels of psaB, rbcL and SAG12 were similar as in the control plants. However, in the course of recovery typical chloroplast senescence symptoms were observed only in individually darkened leaves while re-irradiated DP leaves maintained high photosynthetic capacity.

The function of SNF2 ATPases, the major catalytic subunits of chromatin remodeling complexes, in plants is not sufficiently understood. Here we identified 39 putative SNF2 genes of rice (Oryza sativa L.) by homology analyses and analyzed the expression profiles of eight of them in response to phytohormones and abiotic stresses. Our results indicated that expression of the SNF2 genes was affected by auxin, gibberellin, cytokinin, abscisic acid, ethylene, and some abiotic stresses such as heat, chilling, darkness, drought and salinity. It suggests that, like Arabidopsis SNF2s, rice SNF2 proteins may function in phytohormone signaling pathways and/or be associated with the resistance to abiotic stresses, but in distinct manners from their Arabidopsis orthologs. Some SNF2 proteins in rice may be involved in cross-talk of the signaling pathways between phytohormones and abiotic stresses.

Supply of aqueous solution of triadimefon (20 mg dm^-3) to unstressed green gram plants increased the contents of soluble proteins, amino acids, nitrate and nitrite, and the activity of nitrate reductase in the leaves and nitrate reductase in nodules. The nitrogenase activity in nodules and roots was also increased. Number and fresh mass of nodules and their nitrate and nitrite contents were also higher than those of the controls. In contrast, the UV-B stress (12.2 kJ m^-2 d^-1) suppressed nodulation and nitrogen metabolism in leaves and roots compared to plants under natural UV-B (10 kJ m^-2 d^-1). Triadimefon-treated plants did not show such severe inhibitions after exposure to elevated UV-B. Thus triadimefon increased their tolerance to UV-B stress.

A reproducible in vitro regeneration system for Nepalese kutki (Picrorhiza scrophulariiflora Pennell) was developed from in vitro leaf derived callus. Induction of more than seven shoot buds per explant was achieved on Woody plant medium (WPM) supplemented with 0.53 μM α-napthaleneacetic acid (NAA) and 0.23 μM kinetin (KIN). The shoots were elongated on WPM supplemented with 0.44 μM 6-benzylaminopurine (BAP) and rooted on WPM supplemented with 5.3 μM NAA within 2 weeks. The random amplified polymorphic DNA (RAPD) analysis indicated genetic uniformity of the micropropagated plants with its donor plants.

Lettuce plants were treated with gibberellic acid (GA₃) and uniconazole (UZ; a gibberellin synthesis inhibitor) to investigate the influence of GA₃ on cell division frequency in the shoot apical meristem (SAM) during stem elongation and flower initiation in lettuce (Lactuca sativa) grown in a greenhouse. GA₃ (0.1 mM) was sprayed on the surface of outer leaves and uniconazole solution (0.86 mM) was applied to the soil. GA₃ increased cell division frequency in the peripheral zone and the rib meristem of shoot apices, and this was associated with the stimulation of stem elongation. UZ treatment decreased cell division frequency in the peripheral zone, rib meristem and subapical pith, and this was associated with restricted stem elongation. Treatment with UZ and GA₃ together induced minor stem elongation. Flower induction occurred 3 d earlier in the GA₃ and UZ+GA₃ treatments than in the control, while the UZ treatment delayed flower initiation for more than 9 d relative to the control.

Strawberry (Fragaria ananassa Duch.) seedlings were pretreated with hexanoic acid 2-(diethylamino)ethyl ester (DA-6) in concentrations of 0, 10, 20 and 40 mg dm^-3 and then subjected to chilling and rewarming. The effects of applied DA-6 on the generation of reactive oxygen species (O₂ ^-, H₂O₂), lipid peroxidation, proline accumulation and photosynthesis were evaluated. Pretreatment with DA-6 alleviated the inhibition of superoxide dismutase (SOD), catalase (CAT) and ascorbate peroxidase (APX) activities caused by chilling stress thus reducing O₂ ^- and H₂O₂ production and lipid peroxidation in pretreated plants. DA-6 pretreatment also accelerated accumulation of proline and reduce the decrease in proline content after rewarming. DA-6 pretreatment increases maximum quantum yield of photosystem 2 (F_v/F_m), actual photochemical efficiency of photosystem 2 (Φ_PS2), photochemical quenching coefficient (qP) and net photosynthetic rate (P_N) and decreases non-photochemical quenching coefficient (qNP) of the seedlings under chilling stress. DA-6 pretreatment also increased the recovery rate of photosynthesis after rewarming.

The allelopathic potential of four tree species on soil microbial populations and some herbaceous plants (two understory species and one general biotest species) was investigated. Effects of three nonindigenous tree species, Eucalyptus globulus Labill, Pinus radiata D.Don and Acacia melanoxylon R.Br., on microorganisms participating in the cycle of nitrogen were evaluated, comparing them with those produced by the autochthonous Quercus robur L. Influence of the trees on Lactuca sativa L., Dactylis glomerata L. and Trifolium repens L. was also checked in bioassays. Cell numbers of Nitrosomonas sp. were negatively affected by Acacia and Eucalyptus stands, mainly during spring, when flowers are especially abundant on the ground. Proteolytic microorganisms were also negatively affected by Eucalyptus and Pinus stands, whilst Quercus stand did not show any toxicity. Soil bioassays showed clear inhibitory effects on germination and growth of understory plants, particularly soils from Eucalyptus and Acacia stands. The greatest effects had the soil from Acacia stand, which was phytotoxic during the whole period of germination and growth of understory plants. Allelopathic phenomena could be, at least partially, responsible of the low species diversity in the understory of the nonindigenous tree stands.

Susceptibility of Mexican cacti to Agrobacterium rhizogenes was evaluated in 65 species of 22 genera. Stem discs taken from in vitro cultured plants were inoculated with Agrobacterium rhizogenes A4 agropine-type strain that contains the wild RiA4 plasmid and the binary vector pESC4 with the nptII and gus genes. Hairy roots were produced directly from wounds, or starting from calli generated on the wounded surface, in 34 of the evaluated species. The frequency of hairy roots formation, the number of roots per explant and its growth rates were variable among the tested species. In the 31 remaining species the production of transformed roots was not observed under the conditions used in these experiments. Histochemical detection of β-glucuronidase (GUS) activity demonstrated the expression of this foreign gene in the hairy roots. PCR analyses demonstrated the presence of the rolB and nptII genes in the DNA of the transformed roots. The patterns of alkaloid-like compounds obtained by thin layer chromatography in some of the tested species were qualitatively similar between the transformed and non-transformed roots.

Three clones of tobacco transformed with the T-DNA of Agrobacterium rhizogenes Ri plasmid A4b cultivated in vitro on a solid agar medium neither showed pronounced morphological diversity nor significantly differed in chlorophyll (Chl) contents from control plants. However, the transformation caused a 27 to 83 % decay in leaf oxygen evolution and in both ribulose-1,5-bisphosphate carboxylase (RuBPC) and phosphoenolpyruvate carboxylase (PEPC) activities. Therefore, the transformation brought about a reduction of active PEPC as well as activable RuBPC amounts in plant tissues. Individual clones did not mutually differ. In tobacco transformed with A. rhizogenes strain TR101 and grown in soil only the mean leaf area tended to reduce. Chl contents, Chl a/b ratio, oxygen evolution, and activities of both RuBPC and PEPC were insignificantly affected by the transformation.

Cytokinin (CK) content and activities of several antioxidant enzymes were examined during plant ontogeny with the aim to elucidate their role in delayed senescence of transgenic Pssu-ipt tobacco. Control Nicotiana tabacum L. (cv. Petit Havana SR1) and transgenic tobacco with the ipt gene under the control of the promoter of small subunit of Rubisco (Pssu-ipt) were both grown either as grafts on control rootstocks or as rooted plants. Both control plant types showed a decline in total content of CKs with proceeding plant senescence. Contrary to this both transgenic plant types exhibited at least ten times higher content of CKs than controls and a significant increase of CK contents throughout the ontogeny with maximal values in the later stages of plant development. Significantly higher portion of O-glucosides was found in both transgenic plant types compared to control ones. In transgenic plants, zeatin and zeatin riboside were predominant type of CKs. Generally, Pssu-ipt tobacco exhibited elevated activities of antioxidant enzymes compared to control tobacco particularly in the later stages of plant development. While in control tobacco activity of glutathione reductase (GR) and superoxide dismutase (SOD) showed increasing activity up to the onset of flowering and then gradually decreased, in both transgenic types GR increased and SOD activity showed only small change throughout the plant ontogeny. Ascorbate peroxidase (APOD) was stimulated in both transgenic types. The manifold enhancement of syringaldazine and guaiacol peroxidase activities was observed in transgenic grafts throughout plant ontogeny in contrast to control and transgenic rooted plants, where the increase was found only in the late stages. Electron microscopic examination showed higher number of crystallic cores in peroxisomes and abnormal interactions among organelles in transgenic tobacco in comparison with control plant. The overproduction of cytokinins resulted in the stimulation of activities of AOE throughout the plant ontogeny of transgenic Pssu-ipt tobacco.

Pea (Pisum sativum L., cv. Scinado) seedlings were exposed to low doses of ultraviolet-B (UV-B; 4.4 and 13.3 kJ m^-2 d^-1) or UV-C (0.1 and 0.3 kJ m^-2 d^-1) radiation for 14 d. Aminocyclopropane carboxylic acid (ACC), indoleacetic acid (IAA) and abscisic acid (ABA) contents were quantified by gas chromatography coupled to mass spectrometry (GC-MS). The accumulation of ACC upon irradiation was dose-dependent. ABA content was reduced and IAA content increased upon UV-C treatment whereas the UV-B doses used did not cause significant changes in ABA and IAA contents.

A rapid and highly-effective method for micropropagation from nodal segment and shoot tip explants was established for Coleus blumei Benth. Nodal segments and shoot tips were inoculated on MS medium containing 0.7 % agar, 3 % commercial sugar, and different combinations of 6-benzyladenine (BA) with indole-3-acetic acid (IAA), indole-3-butyric acid (IBA) or α-naphthaleneacetic acid (NAA). Hundred percent shoot induction from both explants was achieved on the medium containing BA (2 mg dm^-3) and NAA (1 mg dm^-3). Shoot tips were proved to be the better explant in comparison to nodal segments in having high rate of shoot induction and more number of shoots. The same media conditions were found suitable for shoot multiplication. Multiplied shoots rooted best on MS medium supplemented with IBA (2 mg dm^-3). Micropropagated plants were successfully established in soil after hardening, with 100 % survival rate.

A reproducible protocol has been developed for high frequency plant regeneration from immature embryos of Argyrolobium roseum Jaub & Spach, an important medicinal legume. Green nodular calli were initiated from immature embryos excised from 10-d-old pods in 70 % of cultures within 3 weeks when grown on Murashige and Skoog (MS) medium supplemented with 0.5 mg dm^-3 benzylaminopurine (BAP) + 0.25 mg dm^-3 indole-3-acetic acid (IAA). Subsequent transfer of 5 mm² callus pieces to MS medium supplemented with BAP (0.5 mg dm^-3) alone or in combination with IAA (0.25 mg dm^-3) facilitated regeneration of multiple shoots. Organogenic calli bearing multiple shoots when transferred to MS medium supplemented with BAP (0.5 mg dm^-3) + IAA (0.25 mg dm^-3) supported rapid shoot elongation. Shoot propagules subcultured to Gamborg's medium (B5) with 0.5 mg dm^-3 indole-3-butyric acid (IBA) rooted with 80 % frequency and developed into phenotypically normal plants. Plantlets were successfully acclimatized in a sterile mixture of sand and garden soil (1:1) under greenhouse and thereafter transferred to field beds.

Genetic engineering of rice (Oryza sativa L. cv. Pusa basmati 1) using synthetic Cry1Ac gene has been achieved by "particle bombardment". Scutellar tissues excised after 5 - 6 d from mature seeds cultured on induction medium were bombarded using gold particles coated with a mixture of Cry1Ac and marker genes on medium with osmoticum. Bombarded tissues were subjected to 30 mg dm^-3 hygromycin selection for two cycles. The selected calli after GUS assay were transferred to shoot regeneration medium. Regenerated shoots were rooted and plantlets (T₀) were grown to full maturity. Polymerase chain reaction (PCR) analysis of T₀ plants using Cry1Ac specific primers revealed the presence of Cry1Ac gene in 65 % plants. Phenotypic assay, β-glucuronidase assay and PCR during T₁ generation revealed the inheritance of the Cry1Ac and marker genes along with the native plant genes.

In red clover (Trifolium pratense L.) four unique cDNAs encoding phenylalanine ammonia lyase (PAL, EC 4.3.1.5) were identified (PAL1-4). PAL2-4 encode nearly identical proteins (> 97 %) that are only 89 % identical to that encoded by PAL1. Under normal growing conditions, in young leaves and flowers PAL1 mRNA accumulates to higher levels than that of PAL2-4 whereas in mature leaves and stems, mRNA levels are similar for PAL1 and PAL2-4. Treatment of red clover seedlings with yeast elicitor preparation results in an approximately six-fold induction of PAL2-4 transcripts within 1 h of treatment but only a modest induction of PAL1 transcripts. These results suggest that while both classes of enzymes play a role in biosynthesis of phenylpropanoid compounds under normal growing conditions, PAL2-4 enzymes are also involved in pathogen defense responses.

Multiple shoot buds could be induced directly from internode explants of Celastrus paniculatus inoculated on Murashige and Skoog's (MS) medium containing different growth regulators. The best response was obtained when 4.44 μM 6-benzylaminopurine (BAP) was incorporated in the medium. Incorporation of indole-3-acetic acid (IAA) and α-naphthalene acetic acid (NAA) did not improve response, rather promoted callusing. Adventitious shoot buds could be multiplied and elongated on MS medium containing 2.22 μM BAP. Rooting of shoots (80 %) was obtained when their bases were dipped in pre-autoclaved indole-3-butyric acid (IBA) solution (2.45 mM) for 10 min followed by their implantation on medium containing 1/4 MS salts, 1.0 % sucrose and 0.6 % agar. Out of 500 plantlets subjected to hardening, 410 were successfully hardened under greenhouse conditions. Twenty plants were established in field while remaining of them were transferred to nursery conditions without any mortality.

The emissions of volatile organic compounds, VOC, from plants have strong relevance for plant physiology, plant ecology, and atmospheric chemistry. We report here on the current knowledge of the many internal (genetic and biochemical) and external (abiotic - temperature, light, water availability, wind, ozone, and biotic - animal, plant and microorganisms interactions) factors that control emission rates of different VOC by altering their synthesis, vapour pressure or diffusion to the atmosphere. The complex net of these factors, their interactions and the different responses of the different VOC produces the large qualitative and quantitative, spatial and temporal variability of emissions and the frequent deviations from current standard emission models. The need for a co-operative multidisciplinary multiscale research to disentangle this complex and important issue of plant VOC emissions is reminded.

The aim of this study was to investigate the protein alterations in the transgenic kiwifruit (Actinidia deliciosa) overexpressing a transcription factor gene, OSH1. Although transgenic plant with introduced OSH1 indicates suppression of gibberellin (GA)-20 oxidase activity, application of GA to transgenic kiwifruit could not completely recover plant morphology and protein profiles. Eleven proteins decreased in the transgenic kiwifruit detected by two-dimensional polyacrylamide gel electrophoresis showed homologies to kiwifruit hypothetical protein, osmotin I and photosynthesis related protein. These results suggest that introduction of an OSH1 into kiwifruit caused wide-range alterations at protein level and alterations of protein accumulation should be considered to evaluate the substantial equivalence of plants transformed by a transcription factor.

Hairy root cultures of Hypericum perforatum were obtained following inoculation of aseptically germinated seedlings with A. rhizogenes strain A4M70GUS. Effect of sucrose on the growth and biomass production of hairy root cultures was investigated. Hairy root cultures spontaneously regenerated shoots buds from which a number of shoot culture clones was established. Transformed shoot cultures exhibited good shoot multiplication, elongation and rooting on a hormone-free woody plant medium. Plants regenerated from hairy roots were similar in appearance to the normal, nontransformed plants.

Rice overexpressed thaumatin-like protein gene and the proteins from the leaf blades of 2-week-old transgenic rice seedlings were fractionated into cytosolic and membrane fractions, and separated by two-dimensional polyacrylamide gel electrophoresis and stained with Commassie brilliant blue. Among of 440 detected proteins, 5 proteins were up-regulated and 5 proteins were down-regulated by the overexpression of thaumatin-like protein. In the sense thaumatin-like protein transgenic rice and/or in rice inoculated with Xanthomonas oryzae pv. oryzae (Xo7435), 2-cys peroxiredoxin, thaumatin-like protein and glycine cleavage H protein were up-regulated, while oxygen evolving complex protein 2 was down-regulated. These results suggest that thaumatin-like protein-mediated disease resistance of rice against bacterial blight disease is the results of changes in proteins related to oxidative stress and energy metabolism in addition to changes in proteins related to defence.

Exposure of barley plants (Hordeum vulgare L.) to soil flooding for 2 to 24 h reduced the net photosynthetic rate and transpiration rate. Stomatal conductance also decreased in flooded plants. Stomatal closure started within 2 - 6 h and stomata remained closed up to 24 h of treatment.

The activities of antioxidative enzymes and contents of proline and total phenolics were assayed in roots of two maize (Zea mays L.) genotypes grown in a medium containing nitrate (NO₃ ^-) or both nitrogen forms, nitrate and ammonium (NH₄ ⁺/NO₃ ^-). An increase in the activities of class III peroxidases (POD), superoxide dismutase (SOD), ascorbate peroxidase (APX), ascorbate oxidase (AO) and proline content, and decrease in phenolic content were observed in NH₄ ⁺/NO₃ ^- in comparison with NO₃ ^- grown plants. When polyethylene glycol (PEG) was added to both nitrogen treatments, the content of total phenolics and proline was increased, especially in NH₄ ⁺/NO₃ ^- treatment. The PEG treatment decreased enzyme activities in NH₄ ⁺/NO₃ ^- grown plants, but in NO₃ ^- grown plants activities of POD and SOD were increased, opposite to decreased APX and AO. Isoelectric focusing demonstrated increased activities of acidic POD isoforms in PEG treated NO₃ ^- grown plants, and lower activities of both, acidic and basic isoforms in NH₄ ⁺/NO₃ ^-grown plants.

Zephyr lily (Zephyranthes grandiflora), an important ornamental plant has been micropropagated in vitro after controlling microbial contamination by a pretreatment with 0.2 % Bavistin and 0.1 % Pantomycin for 4 h before final sterilization with 0.1 % mercuric chloride. In 67 % of the sterile cultures, 11 shoots on average were regenerated directly from basal half of bulb scales in Murashige and Skoog (MS) medium containing 3 % sucrose and 2 mg dm^-3 benzylaminopurine (BAP). Shoots emerged in bunches on a basal achlorophyllous bulbous part. Combination of 2 mg dm^-3 BAP with 1 mg dm^-3 gibberellic acid (GA₃) enhanced shoot growth. Stout roots (maximum of 5-6 per shoot) were developed in presence of 1 mg dm^-3 indole-3-butyric acid (IBA). Micro-bulbs showed potential of regeneration and could be used as secondary explants. The morphologically identical plants derived by in vitro propagation were genetically identical as shown by PCR based ISSR marker analysis of genomic DNA.

In the conversion of oleic acid to linoleic acid, δ¹²-fatty acid desaturase (δ¹²-FAD) is involved. Based on the conserved oligo amino acid residues of the FAD2 genes from other plants, a new full-length cDNA (DiFAD2) encoding a δ¹²-FAD was cloned from Davidia involucrata Baill. Sequence analysis indicated that the DiFAD2 gene had an open reading frame (ORF) of 1 149 bp, coding for 382 amino acids residues of 44.3 kDa, pI of the deduced protein was 8.8. The deduced amino acid sequence of the cloned DiFAD2 showed high identities to those genes of other plant δ¹²-FAD. RT-PCR showed that DiFAD2 was expressed in all tissues and expression was abundant in young stems. Expression of DiFAD2 is not enhanced by low temperature and the altered polyunsaturated fatty acid content in leaves treated with low temperature may be due to the post-transcriptional regulation of the DiFAD2 gene or the other FAD2 gene family regulation.

Secondary embryogenesis from rapeseed microspore-derived embryos (MDEs) was studied in three Brassica napus L. cultivars Global, PF₇₀₄ and Option. The best results in terms of secondary embryogenesis percentage obtained in cultures of Global and PF₇₀₄ MDEs (75.88 and 65.97 %, respectively) and PF₇₀₄ produced the highest number of secondary embryos per each primary embryo (14.91 ± 2.18). After optimization of physical parameters, rapeseed hypocotyls of MDEs were bombarded with microcarriers coated with a plasmid containing GUS reporter gene. The highest levels of transient GUS expression were obtained using bombardment with gold particles of 1.6 µm, at helium pressure of 9.3 MPa, a bombardment distance of 9 cm, chamber vacuum pressure of 7.1 × 10^-6 kPa and single bombardment in bombardment medium containing 0.4 M mannitol.

We determined the cold (freezing) tolerance of five Spanish populations of the perennial shrub Bituminaria bituminosa (L.) C.H. Stirton (Fabaceae), as the temperature at which 50 % of leaf electrolytes are released (LT₅₀) using leaves of field-grown plants, obtained in two winters and one spring. The freezing tolerance was greater in winter and reflected the minimum temperatures at the original sites from which the populations were obtained. Tolerance in vitro was related to osmotic adjustment in the leaves; more negative osmotic potential values and more positive pressure potential values (MPa) were associated with greater tolerance. Tolerance and osmotic potential were not related to leaf cation contents but to leaf amino acids, soluble sugar and proline contents.